SUMMARY The National Eye Institute has identified the restoration of vision as an ?audacious goal? requiring the reestablishment of neural connections from a functional retina with the brain. Unfortunately, the regeneration of adult mammalian CNS axons is extremely limited or nil. The reconnection of retinal ganglion cells with lateral geniculate neurons to support vision recovery requires overcoming endogenous limitations on axonal regeneration. A small group of genes limiting regeneration in particular retinal ganglion cell subtypes have been identified, but only a very small fraction of the genome has been tested with regard to a role in limiting axon regeneration. We hypothesize that additional regeneration-regulating factors exist, and that conservation of function across cell types, species and modes of growth will facilitate their discovery. We have completed a genome-wide shRNA-based screen for endogenous genes limiting the repair of axons in the mammalian CNS using cultured mouse cerebral cortex neurons with over 135,000 separate regeneration experiments. Across 17,000 genes, this loss of function screen yielded 500 regeneration genes. Here, our primary goal is to test this selected list for the ability to promote retinal ganglion cell axon regeneration in vivo. We expect that genes with the most robust regeneration-limiting function will have conserved function from cortical neurons to retinal ganglion cells. Amongst the cortical axon regeneration gene list, we will prioritize genes for optic nerve regeneration experiments using two additional datasets. In one experiment, we determined the distinct transcriptional profiles of sprouting versus non-sprouting mouse CST neurons after CNS injury, hypothesizing that the most relevant genes will participate in both sprouting and regeneration, as is the case for previously described regeneration factors. We have also conducted experiments to identify species conservation of regeneration by screening for genes that regulate motor axon regeneration in the nematode C. elegans. Factors common to multiple experimental systems are expected to identify fundamental mechanisms in regeneration that are likely to affect the equivalent process in human visual system. For those newly discovered genes whose loss of function supports optic nerve regeneration, secondary studies will assess synergy amongst positive genes, retinal ganglion cell type specificity of action, and central axonal pathfinding function. This project builds on genetic screens in the mature mouse central nervous system and C. elegans to analyze novel mechanisms that promote axon regeneration after mammalian retinal ganglion cell axotomy.